A novel approach is described for the detection of soluble aggregates of deoxyhemoglobin S in dilute solution. It is based on the binding of pteroylheptaglutamate (PteGlu7), which distinguishes between unpolymerized and polymerized hemoglobin tetramers. The fraction of PteGlu7) bound will be measured at constant PteGlu7 concentration (1x10 minus 6M) in the presence of increasing hemoglobin concentrations in large excess. Under these conditions the ratio of bound to free PteGlu7 is directly proportional to the concentration of unpolymerized hemoglobin tetramers. The measurements will be carried out by anaerobic ultrafiltration using 14C labeled PteGlu7. The investigations will be divided into: (1) Soluble aggregates as a function of Hb concentration below the minimum geling concentration and (2) Pregelation nuclei as a function of time during the delay period preceding gelation. The method will be used to explore the conditions influencing the formation of pregelation aggregates and to characterize the intermolecular bonds involved. This will include the effect of temperature, pH, ionic strength, etc., as well as the use of chemically modified and mutant hemoglobins, the effect of other rhemoglobins, which solubilize HbS, such as HbF and HbA2, and, especially the influence of antisickling agents. It is hoped that this study could lead to new strategies from a therapeutic point of view, since it is possible that the sickling process could be interrupted not only by preventing aggregation, but also by actually stabilizing the soluble aggregates so as to prevent their further polymerization into fibers.